Neodymium oxide

Endpoint summary

Description of key information

REPEATED DOSE TOXICITY: ORAL

NOEL male 300 mg/kg bw/day, rat (haematological and clinical chemistry changes); NOAEL male 1000 mg/kg bw/day, rat; NOAEL female 300 mg/kg bw/day, rat (a reduction in bodyweight gain during the lactation period, haematological changes and clinical chemistry changes at 1000 mg/kg bw/day), OECD 422, Charles River (2013).

REPEATED DOSE TOXICITY: INHALATION

No study available, a data waiver has been submitted to address this endpoint.

REPEATED DOSE TOXICITY: DERMAL

No study available, a data waiver has been submitted to address this endpoint.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 September 2012 - 7 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: On dispatch from the supplier, the animals were all approximately 7 - 8 weeks old. At initiation of dosing, the animals were approximately 9 - 10 weeks old.
- Weight at study initiation: On dispatch from the supplier, a sample of the males weighed 250 - 275 g and a sample of the females weighed 192 - 215 g. At initiation of dosing, the animals weighed 340 - 401 g for males and 212 - 285 g for females.
- Housing: Animals were housed in cages, suspended on a series of racks. Male and female cages were racked separately.
Animals were housed in polycarbonate cages with stainless steel grid tops and solid bottoms, with approximate dimensions of 61 x 43.5 x 24 cm. Cages were suspended on moveable racks and fitted with water bottles and integrated stainless steel food hoppers.
The animals were initially housed 2 or 3 per cage. A few days prior to pairing for mating, males were transferred to individual cages with a stainless steel grid insert. After mating, the males were re-housed with their original cage mates.
Mated females were transferred to individual solid bottom cages. White paper tissue was supplied as nesting material from Day 20 of gestation.
- Diet (e.g. ad libitum): Rat and mouse breeder diet was provided ad libitum.
- Water (e.g. ad libitum): Water taken from the public supply was available ad libitum.
- Acclimation period: 13 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): A minimum of 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): A 12 hour light/dark cycle was in operation beginning at 07:00 h.

IN-LIFE DATES: From: 4 September 2012 To: 2 November 2012

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1 % w/v

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4 °C, and dispensed daily. They were prepared by adding an appropriate amount of vehicle to the required amount of test material and were mixed by magnetic stirring and high shear mixer until a homogenous solution was obtained. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes prior to dosing and continuously during dosing.
All formulations were used within the 8 day stability period that was established previously at the testing laboratory.

VEHICLE
- Concentration in vehicle: 10, 30 or 100 mg/mL for the 100, 300 and 1000 mg/kg/day dose levels, respectively.
- Dose volume: 10 mL/kg. The volume administered to each animal was determined on each day by the weight of that animal as measured at the time of administration, except during late gestation; from Day 16 of gestation until when parturition was complete, the dose volume was determined by the weight of the animal on Day 16 of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYTICAL METHOD
Analyses were performed by ICP-Optical Emission Spectroscopy using a validated analytical procedure. Samples to be analysed were transferred at ambient temperature to the analytical laboratory and stored in a refrigerator set to maintain 4 °C.

CONCENTRATION AND HOMOGENEITY ANALYSIS
From each formulation, duplicate sets of top, middle and bottom samples (ca 0.5 mL) were taken at each sampling time point (all concentrations on weeks 1 and 4) and sent to the analytical laboratory for analysis (duplicate middle samples only obtained from control formulations). Additional triplicate samples from the top, middle and bottom (middle sample only from control) were also taken as back up samples. The results of the sample concentration were considered acceptable if they were within ± 10 % of theoretical concentration. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentration of ≤10 % for each group.

The results of the analyses of dosing formulations prepared for use on Day 1 of dosing and Week 4 of dosing were all found to be within the ± 10 % acceptance criteria. A low coefficient of variation was obtained (<5.2 %), indicating that the formulations were homogenous. These results show an acceptable accuracy of formulation for the duration of the study.

Duration of treatment / exposure:

The males were dosed for 4 weeks, starting 2 weeks prior to mating. The females were dosed 2 weeks prior to mating, throughout mating, gestation and through to at least day 4 of lactation.
Several females were not dosed on their respective days of parturition due to the animals starting to give birth prior to the commencement of dosing on that day.

Frequency of treatment:

Once daily.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were agreed after a review of existing relevant toxicological data, including a 14 day dose range finding study conducted at the testing facility in which dose levels up to 1000 mg/kg/day produced no adverse reaction to treatment.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/Moribundity checks on all animals were carried out early morning and as late as possible each day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pre-trial, all animals received a detailed clinical examination, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta. All animals were examined for reaction to treatment daily during the course of the study. The onset, intensity and duration of any signs were recorded.

DETAILED FUNCTIONAL OBSERVATIONS (PARTIAL EXAMINATIONS)
Once during the pre-treatment period (week -1) and weekly thereafter, a more detailed examination was made on all animals.
- Cageside Observations: prostration, lethargy, writhing, circling, breathing abnormalities, gait abnormalities, tremor, fasciculation, convulsions, biting (of cage components or self mutilating), vocalisations, piloerection, ease of removal from the cage, body temperature, condition of the coat, presence of salivation and overall ease of handling.
- Condition of the eyes, checked for: pupillary function (reaction to visual stimulus), miosis, mydriasis, exophthalmos, encrustation, lacrimation.
Observations in a standardised area (2 min observation): latency (time to first locomotory movement), level of mobility, rearing, grooming, urination/defecation, arousal (level of alertness), posture (tremor/convulsions, vocalisation, piloerection), palpebral closure, gait abnormalities and stereotypy (excessive repetition of behaviours) and/or unusual behaviours.

BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweights were recorded one week prior to the start of treatment. From the start of treatment, the individual bodyweights were recorded daily.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was measured for both sexes weekly, starting 1 week prior to dosing until pairing for mating. After pairing, the female food consumption was measured over Days 0 - 7, 7 - 14 and 14 - 20 of gestation and Days 0 - 4 of lactation. Male food consumption did not recommence after pairing for mating.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examinations were taken pre-trial for all animals, in Week 4 for all male animals and shortly prior to sacrifice for all female animals.
The eyes were examined using an indirect ophthalmoscope after the application of a mydriatic agent (1 % Tropicamide, Mydriacyl). The following areas were evaluated: anterior, lenticular and fundic areas.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples were obtained during Week 4 for males and on or after Day 4 of lactation for females. Samples were collected via the tail vein after careful cleaning of the sample site to avoid any possible contamination. 0.5 mL of blood was then transferred into plastic tubes containing EDTA as an anticoagulant.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 5 per group; males, the first 5 animals per group and females the first 5 animals in each group to rear their litter to Day 3 of lactation.
- Parameters evaluated: red blood cell count, haemoglobin concentration, haematocrit, mean corpuscular volume, red blood corpuscular distribution width, mean corpuscular haemoglobin concentration, mean cell haemoglobin, reticulocyte count (absolute), platelet count, white blood cell count, neutrophil count (absolute), lymphocyte count (absolute), monocyte count (absolute), eosinophil count (absolute), basophil count (absolute), large unclassified cells and other cells (as appropriate).
A blood smear was prepared from each haematology sample and was evaluated.

COAGULATION: Yes
- Time schedule for collection of blood: Samples were obtained during Week 4 for males and on or after Day 4 of lactation for females. Samples were collected via the tail vein after careful cleaning of the sample site to avoid any possible contamination. Blood samples (0.9 mL) were taken into tubes containing 0.1 mL trisodium citrate (3.8 % (w/v)). The final sample volume was as close as possible to 1.0 mL to give a final concentration of 0.38 % (blood to citrate ratio of 9:1). The citrated blood samples were centrifuged and the plasma separated into plastic tubes and analysed.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 5 per group; males, the first 5 animals per group and females the first 5 animals in each group to rear their litter to Day 3 of lactation.
- Parameters evaluated: activated partial thromboplastin time, fibrinogen and prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Samples were obtained during Week 4 for males and on or after Day 4 of lactation for females. Samples were collected via the tail vein after careful cleaning of the sample site to avoid any possible contamination. 1.0 mL of blood was transferred into tubes containing lithium heparin and the samples centrifuged. The plasma was then examined for the appropriate parameters.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 5 per group; males, the first 5 animals per group and females the first 5 animals in each group to rear their litter to Day 3 of lactation.
- Parameters evaluated: urea, glucose, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatine phosphokinase, lactate dehydrogenase, sodium, potassium, chloride, total protein, albumin, globulin, albumin/globulin ratio, cholesterol, creatinine, total bilirubin, calcium, inorganic phosphate.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
FUNCTIONAL TESTS (FULL EXAMINATION)
The following additional functional assessments were performed on 5 males per group during Week 4 and on 5 females per group during lactation.
Reaction to sudden sound (click above the head), reaction to touch on the rump with a blunt probe, grip strength, pain perception, landing foot splay, motor activity and any other abnormality not already recorded in the above screening battery.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Terminal Procedures: The males were killed when mating was completed and the animals had been dosed for at least 4 weeks (Day 30). The females and litters were killed between Day 5 and 7 of lactation. Animals of 10 days of age were killed by exposure to carbon dioxide followed by exsanguination. Animals less than 10 days of age were killed by intra-peritoneal injection of sodium pentobarbitone.
- Necropsy: All adult animals were subjected to a complete necropsy examination which included evaluation of the carcass and musculoskeletal system, all external surfaces and orifices, cranial cavity and external surfaces of the brain and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tracts of all females were examined.
The following organs were weighed at necropsy for all adult animals before sampling and preservation: brain, epididymis, adrenal gland, pituitary gland, prostate gland, thyroid gland, heart, kidney, liver, lung, ovary, spleen, testis, thymus and uterus.

HISTOPATHOLOGY: Yes
Representative samples of the following tissues were collected from all adult animals and preserved as appropriate: animal identification (microchip), aortic artery, bone marrow smear, bone marrow (femur and sternum), femur (bone), rib (bone), sternum (bone), brain, cervix, epididymis, eye, adrenal gland, harderian gland, lacrimal gland, mammary gland, parathyroid gland, pituitary gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, large intestine (caecum, colon and rectum), larynx, liver, lung, lymph node (mandibular and mesenteric), skeletal muscle, nasal cavity, optic nerve, sciatic nerve, oesophagus, ovary, oviduct, pancreas, pharynx, skin, small intestine (duodenum, ileum and jejunum), spinal cord, spleen, stomach, testis, thymus, tongue, trachea, ureter, urinary bladder, uterus and vagina.
Histopathological evaluation of all tissues were undertaken for the 5 selected males and females in the Control and High dose groups (the same animals that were used for the laboratory investigations) and for the kidneys only from all remaining females and from all males in group 1 and 4.

Other examinations:

As part of the reproductive / developmental portion of the study, observations were also carried out on mating procedure, reproductive and viability indices were calculated, females with litters were observed during lactation and at the end of the study, pups were sacrificed and necropsied.

Statistics:

Where required to assist with interpretation, tests were applied to determine the statistical significance of observed differences between control and groups receiving test material. Unless otherwise stated, all statistical tests were two-sided and performed at the 5 % significance level using in-house software. Pairwise comparisons were only performed against the control group.

Selected bodyweight and food consumption data, haematology, coagulation, clinical chemistry and selected FOB and motor activity data was analysed for homogeneity of variance using the ‘F Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test; i.e., pairwise comparisons were made only if the overall F test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).

Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate. Organ weights were also expressed as a percentage of terminal body weight and analysed using ANOVA as detailed above.

Incidence data was analysed as proportions in a Kruskal-Wallis analysis, or by categorical methods using contingency tables with the Fisher’s Exact Probability test or the Chi-squared test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results".

Mortality:

mortality observed, treatment-related

Description (incidence):

see "Details on results".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results".

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results".

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

see "Details on results".

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results".

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY CLINICAL OBSERVATIONS
There were no premature deaths on this study.
Treatment at levels up to 1000 mg/kg/day in males produced no clinical observations that were considered related to administration of the test material.
At 1000 mg/kg/day in females there was an increase in the number of animals walking on tip toes (between days 12-20) and a slight increase in incidence of animals with irregular respiration (between days 12-14) and hunched posture (between days 12-23), when compared to controls. The effects were transient on each occasion lasting up to 4 hours post dose. The incidences were outside of the historical control range on the equivalent study days.
Treatment at levels up to 300 mg/kg/day in females produced no clinical observations that were considered related to administration of the test material.

BODYWEIGHTS AND BODYWEIGHT CHANGES
At levels up to 1000 mg/kg/day the group mean body weight gain for males was similar to controls.
At levels up to 1000 mg/kg/day the group mean body weight gain for females prior to mating and throughout gestation were similar to controls.
At 300 mg/kg/day and above there was a dose related decrease in female group mean bodyweight gain during the lactation period only. At 1000 mg/kg/day there was a 63 % reduction and at 300 mg/kg/day there was a 26 % reduction in female group mean body weight gain over Days 1 - 4 of lactation, when compared to controls (Table 1). At 300 mg/kg/day the reduced weight gain was still within the mean historical control range and as the group mean bodyweight for animals dosed at 300 mg/kg/day was higher than for other groups, the significance of this reduction is not clear.

FOOD CONSUMPTION
Group mean food consumption prior to pairing for mating was similar to controls in both males and females.
The group mean food consumption in all treated females was similar to controls during gestation and lactation.

OPHTHALMIC EXAMINATIONS
There were no ophthalmoscopy findings which were considered to be related to administration of the test material. All findings were considered to be typical of rats of the age and strain used.

FUNCTIONAL OBSERVATION BATTERY PARAMETERS
Neurotoxicity Clinical Observations
In males there were no neurotoxicity clinical observations during the course of the study that were considered to be related to administration of the test material.
In females there was an increase in the incidence of body held low or hunched in the arena and partial palpebral closure during Weeks 5 and 6 in animals dosed at 1000 mg/kg/day; these values were outside of the historical control range.

Detailed Functional Observations
In males detailed functional observations in treated animals were similar to controls throughout the course of the study. Any slight intergroup differences were considered to be too minor in magnitude to be attributed to the test material.
In females there was a reduction in sectors crossed during Weeks 4 and 6 and number of rears noted in the arena during Week 6 in animals dosed at 1000 mg/kg/day; these values were outside of the historical control range.

Motor activity
There were no differences in motor activity noted between the treated groups and controls that were considered to be related to administration of the test material.

HAEMATOLOGY AND COAGULATION
There was a slight but statistically significant decrease in group mean red blood cell count, haematocrit and haemoglobin (females only) in animals dosed at 1000 mg/kg/day during Week 4 for males and Day 4 of lactation for females. These changes were also seen at 300 mg/kg/day in females (selected parameters are summarised in Table 2). These changes were within the mean historical range for males, but not for females.

CLINICAL CHEMISTRY
In males during Week 4 there were statistically significant decreases in total protein and albumin and an increase in phosphate at 1000 mg/kg/day. Glucose was increased when compared to controls at ≥ 300 mg/kg/day. With the exception of glucose and phosphate levels at 1000 mg/kg/day, these changes were within the mean historical range.
In females there was an increase in alkaline phosphatase at 1000 mg/kg/day when compared to controls (which was also outside of the historical control range). Decreased lactate dehydrogenase and creatine phosphokinase were noted at ≥100 mg/kg/day when compared to controls, but there was no clear dose response relationship and values were within the mean historical control range for creatine phosphokinase In addition, high creatinine, urea and total bilirubin was noted in one female (No. 73F) dosed at 1000 mg/kg/day resulting in a slight increase in group mean values for these parameters compared to controls (selected parameters are summarised in Table 3).

GROSS PATHOLOGY
No test material-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test material.

ORGAN WEIGHTS
No test material-related organ weight changes were noted. There were isolated organ weight values that were different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to administration of the test material.

HISTOPATHOLOGY
There was a treatment associated finding in the kidney at 1000 mg/kg/day, where marked bilateral cortical tubular necrosis and mild diffuse cortical basophilic tubules (bilateral), were recorded in female 73, and mild diffuse cortical basophilic tubules (bilateral) were noted in female 78. The background historical control data shows that this finding has not been seen in lactating female rats on any previous studies. Examination of the kidneys from all females in the mid- and low-dose groups and from all of the males did not reveal any abnormalities.
The higher plasma levels of creatinine and urea recorded in female 73 supported the renal pathology (there were however no microscopic correlates associated with the higher levels of alkaline phosphotase and bilirubin noted in female 73).
The kidney findings are summarised in Table 4.
Otherwise the microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: clinical chemistry

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects observed

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: clinical chemistry, bodyweight and neurotoxicity

Critical effects observed:

not specified

Table 1 Female Bodyweights (g): Group Mean Values During Gestation and Lactation ± Standard Deviation

Dose Level (mg/kg/day)	Day of Gestation					Weight Gain Days 0 - 20	Weight Gain as % of Control	Day of Lactation*
	0	7	14	16	20			1	4
0	268 ± 20	303 ± 22	340 ± 25	361 ± 25	411 ± 32	143	-	280 ± 27	307 ± 28
100	264 ± 9	296 ± 13	334 ± 10	358 ± 11	416 ± 15	152	106	293 ± 9	321 ± 13
300	283 ± 14	317 ± 16	361 ± 23	383 ± 26	447 ± 30	164	115	313 ± 25	333 ± 22
1000	266 ± 22	306 ± 27	348 ± 28	369 ± 28	429 ± 32	163	114	316 ± 35	326 ± 36

*Animals with litters surviving to Day 4 or after only

 

Table 2 Selected Haematology Parameters: Group Mean Values

Sex	Dose Level (mg/kg/day)		Hb (g/dL)	RBC (x 10¹²/L)	Hct (L/L)
Males (Week 4)	0	Mean SD N	15.8 0.7 5	8.91 0.42 5	0.486 0.018 5
	100	Mean SD N	16.1 0.5 5	8.93 0.25 5	0.495 0.012 5
	300	Mean SD N	15.7 0.7 5	9.07 0.53 5	0.483 0.023 5
	1000	Mean SD N	15.2 0.2 5	8.38* 0.21 5	0.462* 0.007 5
Females (Day 4 of Lactation)	0	Mean SD N	13.4 0.2 5	7.12 0.23 5	0.408 0.011 5
	100	Mean SD N	12.6 0.8 5	6.54 0.47 5	0.387 0.028 5
	300	Mean SD N	12.1* 1.1 5	6.42* 0.63 5	0.374* 0.034 5
	1000	Mean SD N	11.2*** 0.8 5	6.07** 0.58 5	0.343*** 0.022 5

Hb = Haemoglobin; RBC = red blood cells; Hct = Haematocrit

Significantly different from the controls: * = p<0.05; ** = p<0.01; *** = p< 0.001

 

Table 3 Selected Clinical Chemistry Parameters: Group Mean Values

Sex	Dose Level (mg/kg/day)		ALP (U/L)	LDH (U/L)	Glu (mmol/L)	T.Bil (µmol/L)	TP (g/L)	Alb (g/L)	Phos (mmol/L)	Crea (µmol/L)
Males (Week 4)	0	Mean SD N	205 32 5	249 64 5	7.57 0.42 5	1.7 0.0 5	68 3 5	43 2 5	2.30 0.27 5	33 2 5
	100	Mean SD N	200 35 5	272 87 5	7.72 0.82 5	1.7 0.0 5	70 4 5	44 2 5	2.43 0.20 5	32 2 5
	300	Mean SD N	184 24 5	252 51 5	8.48* 0.89 5	1.7 0.0 5	67 1 5	43 1 5	2.42 0.17 5	33 3 5
	1000	Mean SD N	199 57 5	188 38 5	8.88** 0.43 5	1.7 0.0 5	64* 2 5	41* 1 5	2.82** 0.18 5	31 3 5
Females (Day 4 of Lactation)	0	Mean SD N	86 19 5	384 147 5	5.76 0.38 5	1.8 0.3 5	64 3 5	43 2 5	1.49 0.57 5	45 4 5
	100	Mean SD N	98 21 5	133*** 53 5	6.14 0.52 5	1.7 0.0 5	63 6 5	41 3 5	1.33 0.62 5	40 3 5
	300	Mean SD N	102 29 5	204** 63 5	5.85 0.73 5	1.7 0.0 5	70 3 5	44 2 5	1.91 0.67 5	45 5 5
	1000	Mean SD N	156* 65 5	247* 41 5	5.96 0.93 5	2.1 0.9 5	64 4 5	41 2 5	1.59 0.76 5	50 19 5

ALP = Alkaline Phosphatase; LHD = Lactate Dehydrogenase; Glu = Glucose; T.Bil = Total bilirubin; TP = Total protein; Alb = Albumin;

Phos = Phosphate; Crea = Creatinine.

Significantly different from the controls: * = p<0.05; ** = p<0.01; *** = p< 0.001

 

Table 4 Summary of Histological Findings in the Kidney

Histological Findings	Group Totals
	Males		Females
	Dose Level (mg/kg/day)
	0	1000	0	100	300	1000
N	10	10	10	10	10	10
No abnormality detected	2	5	6	6	6	6
Necrosis, cortical tubular, bilateral Marked	0	0	0	0	0	1
Necrosis, cortical tubular, bilateral Total Incidence	0	0	0	0	0	1
Basophilic tubules, diffuse, cortical Mild Total Incidence	0 0	0 0	0 0	0 0	0 0	2 2
Chronic progressive nephropathy	0	0	0	1	0	0
Basophilic tubules, focal	7	4	1	2	0	2
Tubular casts	2	1	3	0	0	0
Inflammatory cell foci	1	3	1	1	1	0
Mononuclear cell infiltration, pelvic	0	0	1	0	0	0
Pelvic dilation	0	0	0	2	1	0
Mineral Deposits, medullary	0	0	1	0	2	0

N = the number of animals from which the tissue was examined microscopically

 

Conclusions:

In females some changes were noted during Weeks 5 and 6 at 1000 mg/kg/day in both the dose related and neurotoxicty clinical observations (closed eyes, less activity in the arena, hunched posture, walking on tiptoes). In addition a reduction in bodyweight gain was noted during lactation when compared to the other groups. These changes were not noted in males.
There were no histological correlates with the haematology changes seen at 1000 mg/kg/day in males and at ≥ 300 mg/kg/day in females; or with the clinical chemistry changes seen in the males or the increase in alkaline phosphatase or decreased lactate dehydrogenase and creatine phosphokinase seen in treated females.
The higher plasma levels of creatinine and urea recorded in one female supported the renal pathology noted at 1000 mg/kg/day in this female. There were however no microscopic correlates associated with the higher levels of alkaline phosphatase and bilirubin noted in this female.
In conclusion, under the conditions of this study the No Observed Effect Level (NOEL) in males was considered to be 300 mg/kg/day and the No Observed Adverse Effect Level (NOAEL) in males was considered to be 1000 mg/kg/day. In females the No Observed Adverse Effect Level (NOAEL) was considered to be 300 mg/kg/day.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guideline OECD 422.

Three groups of 10 male and 10 female Sprague-Dawley rats of the Crl:CD (SD) strain were dosed once daily, by oral gavage, with the test material at dose levels of 100, 300, or 1000 mg/kg/day. Another group of 10 male and 10 female rats was dosed with the vehicle (1 % w/v carboxymethylcellulose) following the same dosing regimen as the treated animals and was used as control.

The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca. 6 weeks of treatment). 

The following parameters and end points were evaluated: clinical signs, bodyweights, bodyweight changes, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathological examinations.

In females At 1000 mg/kg/day there was an increase in the number of animals walking on tip toes (between Days 12 and 20) and slight increases in the incidence of irregular respiration (between Days 12 and 14) and hunched posture (between Days 12 to 23) when compared to concurrent controls. The effects were transient on each occasion lasting up to 4 h post dose. Furthermore, some changes were noted during Weeks 5 and 6 at 1000 mg/kg/day in both the dose related and neurotoxicty clinical observations (closed eyes, less activity in the arena, body held low or hunched in the arena). In addition a reduction in bodyweight gain was noted during lactation when compared to the other groups. These changes were not noted in males.

There were no histological correlates with the haematology changes seen at 1000 mg/kg/day in males and at ≥ 300 mg/kg/day in females; or with the clinical chemistry changes seen in the males or the increase in alkaline phosphatase or decreased lactate dehydrogenase and creatine phosphokinase seen in treated females.

The higher plasma levels of creatinine and urea recorded in one female supported the renal pathology noted at 1000 mg/kg/day in this female. There were however no microscopic correlates associated with the higher levels of alkaline phosphatase and bilirubin noted in this female.

 

In conclusion, under the conditions of this study the No Observed Effect Level (NOEL) in males was considered to be 300 mg/kg/day and the No Observed Adverse Effect Level (NOAEL) in males was considered to be 1000 mg/kg/day. In females the No Observed Adverse Effect Level (NOAEL) was considered to be 300 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted to GLP and in accordance with the standardised guideline OECD 422. The study was assigned a reliability score of 1 according to the criteria of Klimisch (1997).

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

In accordance with REACH sections 8.6.1 and 8.6.2 the repeat dose toxicity testing should be conducted in the most appropriate route of administration. In accordance with ECHA’s request a 90-day repeated dose oral toxicity study is being conducted to OECD Guideline TG 408 combined with OECD TG 424. The oral route is the most relevant and as such testing via the inhalation route does not need to be conducted.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

In accordance with REACH sections 8.6.1 and 8.6.2 the repeat dose toxicity testing should be conducted in the most appropriate route of administration. In accordance with ECHA’s request a 90-day repeated dose oral toxicity study is being conducted to OECD Guideline TG 408 combined with OECD TG 424. The oral route is the most relevant and as such testing via the inhalation route does not need to be conducted.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

In accordance with REACH sections 8.6.1 and 8.6.2 the repeat dose toxicity testing should be conducted in the most appropriate route of administration. In accordance with ECHA’s request a 90-day repeated dose oral toxicity study is being conducted to OECD Guideline TG 408 combined with OECD TG 424. The oral route is the most relevant and as such testing via the dermal route does not need to be conducted.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

In accordance with REACH sections 8.6.1 and 8.6.2 the repeat dose toxicity testing should be conducted in the most appropriate route of administration. In accordance with ECHA’s request a 90-day repeated dose oral toxicity study is being conducted to OECD Guideline TG 408 combined with OECD TG 424. The oral route is the most relevant and as such testing via the dermal route does not need to be conducted.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guideline OECD 422.

 

Three groups of 10 male and 10 female Sprague-Dawley rats of the Crl:CD (SD) strain were dosed once daily, by oral gavage, with the test material at dose levels of 100, 300, or 1000 mg/kg/day. Another group of 10 male and 10 female rats was dosed with the vehicle (1 % w/v carboxymethylcellulose) following the same dosing regimen as the treated animals and was used as control.

The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca. 6 weeks of treatment). 

 

The following parameters and end points were evaluated: clinical signs, bodyweights, bodyweight changes, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathological examinations.

 

In females at 1000 mg/kg/day a reduction in mean bodyweight gain was noted during lactation, with body weight losses recorded for few individual animals. There was an increase in the number of animals walking on tip toes (between Days 12 and 20) and slight increases in the incidence of irregular respiration (between Days 12 and 14) and hunched posture (between Days 12 to 23) when compared to concurrent controls. The effects were transient on each occasion lasting up to 4 h post dose. Furthermore, some changes were noted during Weeks 5 and 6 at 1000 mg/kg/day in both the dose related and detailed clinical observations in a standard arena (closed eyes, less activity in the arena, body held low or hunched in the arena). These changes were not noted in males. In addition, necropsy findings and histopathology observations, where available, suggest most of the observations could be related to mis-gavage and consequent general deteriorated well-being of specific animals and thus unrelated to the registered substance.

 

There were no histological correlates with the haematology changes seen at 1000 mg/kg/day in males and at ≥ 300 mg/kg/day in females; or with the clinical chemistry changes seen in the males or the increase in alkaline phosphatase or decreased lactate dehydrogenase and creatine phosphokinase seen in treated females.

 

The higher plasma levels of creatinine and urea recorded in one female supported the renal pathology noted at 1000 mg/kg/day in this female. There were however no microscopic correlates associated with the higher levels of alkaline phosphatase and bilirubin noted in this female.

 

In conclusion, under the conditions of this study the No Observed Effect Level (NOEL) in males was considered to be 300 mg/kg/day and the No Observed Adverse Effect Level (NOAEL) in males was considered to be 1000 mg/kg/day. In females the No Observed Adverse Effect Level (NOAEL) was considered to be 300 mg/kg/day.

Inhalation

In accordance with REACH sections 8.6.1 and 8.6.2 the repeat dose toxicity testing should be conducted in the most appropriate route of administration.  In accordance with ECHA’s request a 90-day repeated dose oral toxicity study is being conducted to OECD Guideline TG 408 combined with OECD TG 424.  The oral route is the most relevant and as such testing via the inhalation route does not need to be conducted.

Dermal

In accordance with REACH sections 8.6.1 and 8.6.2 the repeat dose toxicity testing should be conducted in the most appropriate route of administration.  In accordance with ECHA’s request a 90-day repeated dose oral toxicity study is being conducted to OECD Guideline TG 408 combined with OECD TG 424.  The oral route is the most relevant and as such testing via the dermal route does not need to be conducted.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
A single good quality study was available to address this endpoint.

Justification for classification or non-classification

In accordance with criteria for classification as defined in Annex I, Regulation EC No. 1272/2008 (CLP), the test material does not require classification for repeated dose specific organ toxicity.